Altered glycosylation of surface glycoproteins in tumor cells and its clinical application

Appearance of large Asn-linked sugar chains in the surface glycoprotein is one of the most widely found phenomena in malignant cells and is called the Warren-Glick phenomenon. Comparative study of the sugar chain structures of the surface glycoproteins of cultured cells and their malignant transformants revealed that the altered glycosylation is induced by enhancement of N-acetyl-glucosaminyltransferase V. The change seems to be highly correlated with the metastatic potential of tumor cells.     

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.     

Altered glycosylation is induced in both alpha- and beta-subunits of human chorionic gonadotropin produced by choriocarcinoma

The two subunits of human chorionic gonadotropin (hCG) purified from the urine of a patient with choriocarcinoma were successfully separated by SDS-polyacrylamide gel electrophoresis. A comparative study of the oligosaccharides released from the two subunits by hydrazinolysis revealed that altered glycosylation occurs in both subunits and possibly at all four asparagine sites of the choriocarcinoma hCG molecule.     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells

Background

Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.

Methods

Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.

Results

Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.

Conclusions

Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.

General significance

These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

Glycan Residues Balance Analysis - GReBA: A novel model for the N-linked glycosylation of IgG produced by CHO cells

The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.     

Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application

Therapeutic antibodies have revolutionised treatment of some cancers and improved prognosis for many patients. Over half of those available are approved for haematological malignancies, but efficacious antibodies for solid tumours are still urgently needed. Clinically available antibodies belong to the IgG class, the most prevalent antibody class in human blood, while other classes have not been extensively considered. We hypothesised that the unique properties of IgE, a class of tissue-resident antibodies commonly associated with allergies, which can trigger powerful immune responses through strong affinity for their particular receptors on effector cells, could be employed for passive immunotherapy of solid tumours such as ovarian and breast carcinomas. Our laboratory has examined this concept by evaluating two chimaeric antibodies of the same specificity (MOv18) but different isotype, an IgG1 and an IgE against the tumour antigen folate receptor α (FRα). The latter demonstrates the potency of IgE to mount superior immune responses against tumours in disease-relevant models. We identified Fcε receptor-expressing cells, monocytes/macrophages and eosinophils, activated by MOv18 IgE to kill tumour cells by mechanisms such as ADCC and ADCP. We also applied this notion to a marketed therapeutic, the humanised IgG1 antibody trastuzumab and engineered an IgE counterpart, which retained the functions of trastuzumab in restricting proliferation of HER2/neu-expressing tumour cells but also activated effector cells to kill tumour cells by different mechanisms. On-going efficacy, safety evaluations and future first-in-man clinical studies of IgE therapeutics constitute key metrics for this concept, providing new scope for antibody immunotherapies for solid tumours.     

Activated macrophages digest the extracellular matrix proteins produced by cultured cells.

Activated mouse peritoneal macrophages were cultured directly on the extracellular matrix proteins produced by smooth muscle cells $\text{in}\text{vitro}$. The breakdown of the connective tissue proteins to the level of amino acids was followed by observing the release of radioactivity from matrices labelled with [³H]proline. These studies showed that macrophages produce enzymes capable of digesting the matrix and indicated a major role for the macrophage plasminogen activator in this digestion.     

The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by chinese hamster ovary cells

Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine, is reduced by NH(4) (+) concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked alpha(2,6) sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation. (c) 1995 John Wiley & Sons, Inc.     

Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti‐tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti‐tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME—such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell‐based microenvironment therapies—to provide novel ideas for producing breakthroughs in tumour therapy strategies.     

NG2 precursor cells in neoplasia: Functional, histogenesis and therapeutic implications for malignant brain tumours

Diffusely infiltrating astrocytic tumours of the central nervous system (CNS) are the most frequent intracranial neoplasms and account for more than 60% of all primary brain tumours in man. Until recently, it was generally accepted that the glial component of the mature CNS, consisted of differentiated astrocytes, ependymal cells, oligodendrocytes and the non-neuro-ectodermal microglial cells. There exists a recently recognised population of glial cells that express the NG2 proteoglycan (NG2 cells). NG2 cells are dynamic and undergo rapid morphological changes in response to a variety of CNS pathologies. They are highly motile cells, which interact with various extracellular matrix (ECM) in association with the integrin receptors. During angiogenesis and response to tissue injury, NG2 precursor cells are recruited to sites where vessel growth and repair are occurring. NG2 is over-expressed by both tumour cells and pericytes on the blood vessels of malignant brain tumours. The function of NG2 cells in the CNS, and the notion of them as a source of and/or lineage marker for some gliomas are discussed. In addition, their possible role in glioma angiogenesis, proliferation and invasion will be considered as will their value in provision of targets for clinical and pre-clinical therapeutic strategies in brain tumours.     

Gene gun application in the generation of effector T cells for adoptive immunotherapy

We utilized the gene gun to transfect subcutaneous D5 melanoma and MT-901 mammary carcinoma tumors in situ with a granulocyte/macrophage-colony-stimulating factor (GM-CSF) plasmid complexed to gold particles. There was diminished tumor growth following bombardment with GM-CSF plasmid, which was apparent only during the period of administration. Transgenic GM-CSF was produced by the skin overlying the tumors and not by the tumors themselves. GM-CSF plasmid bombardment resulted in increased cell yields within tumor-draining lymph nodes (TDLN) with at least a 12-fold increase in the percentage of dendritic cells (8.9%) compared to controls (0.7%). Secondarily activated TDLN cells from animals transfected with GM-CSF demonstrated enhanced cytokine release (interferon γ, GM-CSF and interleukin-10) in response to tumor stimulator cells compared to controls, and had an increased capacity to mediate tumor regression in adoptive immunotherapy. There was a small, but detectable, non-specific immune adjuvant effect observed with gold particle bombardment alone, which was less than with GM-CSF plasmid. The adjuvant effect of GM-CSF plasmid required peri-tumoral transgene expression since gene bombardment away from the tumor was ineffective. Received: 27 April 1999 / Accepted: 27 August 1999     

Altered glycosylation in cancer: A promising target for biomarkers and therapeutics

Glycosylation is a well-regulated cell and microenvironment specific post-translational modification. Several glycosyltransferases and glycosidases orchestrate the addition of defined glycan structures on the proteins and lipids. Recent advances and systemic approaches in glycomics have significantly contributed to a better understanding of instrumental roles of glycans in health and diseases. Emerging research evidence recognized aberrantly glycosylated proteins as the modulators of the malignant phenotype of cancer cells. The Cancer Genome Atlas has identified alterations in the expressions of glycosylation-specific genes that are correlated with cancer progression. However, the mechanistic basis remains poorly explored. Recent researches have shown that specific changes in the glycan structures are associated with 'stemness' and epithelial-to-mesenchymal transition of cancer cells. Moreover, epigenetic changes in the glycosylation pattern make the tumor cells capable of escaping immunosurveillance mechanisms. The deciphering roles of glycans in cancer emphasize that glycans can serve as a source for the development of novel clinical biomarkers. The ability of glycans in intervening various stages of tumor progression and the biosynthetic pathways involved in glycan structures constitute a promising target for cancer therapy. Advances in the knowledge of innovative strategies for identifying the mechanisms of glycan-binding proteins are hoped to hold great potential in cancer therapy. This review discusses the fundamental role of glycans in regulating tumorigenesis and tumor progression and provides insights into the influence of glycans in the current tactics of targeted therapies in the clinical setting.     

Characterization of the glycosylation status of proteins

Proteins are posttranslationally modified by a number or mechanisms, by far the most significant of which in terms of the molecular mass and functional diversity is glycosylation. The oligosacchande chains added shortly after biosynthesis at the ribosomc. and further modified while the protein is translocated through the intracellular membranes, have multiple affects on transport, localization, stability, plasma membrane expression activity, and antigenicity. Characterization of the glycosylation patterns at each site around the protein is essential for detailed understanding of structure/function relationships and the design of potential therapeutic agents. This can be achieved by a series of Chromatographie and physicochcmical procedures including HPLC, GC MS. NMR, and computer graphics molecular modeling, which culminate in information on monosaccharide sequence and linkage, glycoprotein conformation, and oligosaccharide-to-protein interactions.     

Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma

We analysed the oligosaccharides of a human IgM produced bya human-human-mouse hybridoma at each of its five conservedheavy chain glycosylation sites. Consistent with previous reports,this IgM possesses sialylated oligosaccharides at Asn171, Asn332and Asn395, and high-mannose-type oligosaccharides at Asn402.In contrast to previous reports for human IgMs, we find thatAsn563 is not occupied by oligosaccharide on perhaps 25% ofIgM heavy chains, while occupied Asn563 sites contain both high-mannose-typeand sialylated oligosaccharides. These latter results are consistentwith the glycosylation at Asn563 previously reported for themouse MOPC 104E IgM. We demonstrate that both the human hybridomaIgM and the mouse MOPC 104E IgM are mixtures of pentamers andhexamers, raising the possibility that the unique findings concerningthe glycosylation at Asn563 in this study and the previous studyof the MOPC 104E IgM could be related, at least in part, tothe different packing requirements of the hexameric geometryand the accessibility of oligosaccharides in the hexameric geometryfor processing to complex type. In addition, we used high-pHanion-exchange (HPAE) chromatography, neutral anion-exchangechromatography, fluorophore-assisted carbohydrate electrophoresisand Western blots to compare the oligosaccharide compositionsof the human hybridoma IgM, pooled human serum IgM and two mousemonoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presenceof N-glycolylneuraminic acid (NeuGc) and N-acetymeuraminic acid(NeuAc) at a 2:1 ratio in the oligosaccharides of the humanhybridoma IgM. The presence of both NeuGc and NeuAc complicatesthe interpretation of HPAE chromato-graphs. glycosylation high-pH anion-exchange chromatography human IgM human—mouse hybridoma oligosaccharide     

Appropriate glycosylation of recombinant proteins for human use

One of the commonest and least well understood posttranslational modifications of proteins is their glycosylation. Human glycoproteins are glycosylated with a bewilderingly heterogeneous array of complex N- and O-linked glycans, which are the product of the coordinated activity of enzymes resident in the endoplasmic reticulum and Golgi apparatus of the cell. Glycosylation of proteins is highly regulated and changes during differentiation, development, under different physiological—and cell culture—conditions and in disease. The glycosylation of recombinant proteins, especially those destined for potential administration to human subjects, is of critical importance. Glycosylation profoundly affects biological activity, function, clearance from circulation, and crucially, antigenicity. The cells of nonhuman species do not glycosylate their proteins in the same way as human cells do. In many cases, the differences are profound. Overall, the species most distant to humans in evolutionary terms, such as bacteria, yeasts, fungi, insects and plants—the species used most commonly in expression systems—have glycosylation repertoires least like our own. This review gives a brief overview of human N- and O-linked protein glycosylation, summarizes what is known of the glycosylation potential of the cells of nonhuman species, and presents the implications for the biotechnology industry.